The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In ...the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal α-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.
To examine the direct effects of tributyltin acetate (TBTA) on osteoclasts and osteoblasts, teleost scale, which has both osteoclasts and osteoblasts and is similar to mammalian membrane bone, was ...used in the present study. The activities of tartrate-resistant acid phosphatase and alkaline-phosphatase, as respective indicators of activity in both cells, were used. In freshwater teleost (goldfish) and marine teleosts (nibbler and wrasse), the osteoclastic activity in the scales did not change as a result of TBTA treatment (10
−
9
to 10
−
5
M). However, the osteoblastic activity decreased in the goldfish, nibbler, and wrasse after 6 h of incubation. In goldfish, even 10
−
10
M of TBTA significantly inhibited the osteoblastic activity. The inhibitory activity in goldfish was stronger than that in nibbler and wrasse. Therefore, details of the mechanism were examined using goldfish. The mRNA expressions of the estrogen receptor and insulin-like growth factor-I, which participate in osteoblastic growth and differentiation, decreased in the TBTA-treated scales. However, the mRNA expression of metallothionein (MT), a metal-binding protein that protects the organism from heavy metal, increased much less than those of cadmium and methyl-mercury. Furthermore, we showed that the plasma calcium and hypocalcemic hormone (calcitonin) level increased in goldfish kept in water containing TBTA (10
−
10
and 10
−
8
M). The current data are the first to demonstrate that, in teleosts, TBTA inhibits osteoblastic activity without affecting osteoclastic activity and disrupts the calcium metabolism, including the calcemic hormone, in goldfish.
Due to the risk of infection from COVID-19, first-year students at Tokyo Medical and Dental University were unable to attend an entrance ceremony and have largely been staying at home since April. ...First-semester classes have been conducted mostly online since the end of Golden Week. We report on the educational effect of face-to-face classes of Chemistry Experiment held from August 4-7 and 11-12 during the second wave of COVID-19. In particular, we introduce as many comments as possible from students in order to emphasize the importance of face-to-face classes.
We examined the difference between conducting experiments on metal ions alone and in pairs in chemistry experiments. Although there was no significant difference in progress when the experiments were ...performed in pairs, a small increase in progress was seen from an increase in the detection rate of some ions. There was little effect of the order of the face-to-face and on-demand experiments.
Advanced glycation end products (AGEs), which are produced by the binding of sugars to proteins, are important factors that cause damage to cells and tissues. AGEs have recently attracted attention ...in the field of anti-aging. In AGEs, glycation intermediates having an α-dicarbonyl structure form crosslinks in the protein, which reduces the function of the protein. In this study, 1-phenyl 1,2-propanedione (PPD) having an α-diketone structure was used as a model compound, and various indole compounds including melatonin were investigated by the method of measuring the AGE cross-linking cleavage action. As a result, it was found that melatonin, serotonin, tryptamine, and 5-methoxytryptamine have an AGE cross-linking cleavage action. It is suggested that these bioamines prevent the production of AGEs and may be effective for anti-aging.
Many recombinant proteins developed or under development for clinical use are glycoproteins, and trials aimed at improving their bioactivity or pharmacokinetics in vivo by altering specific glycan ...structures are ongoing. For pharmaceuticals of glycoproteins, it is important to characterize and, if possible, control the glycosylation profile. However, the mechanism responsible for the regulation of sugar chain structures found on naturally occurring glycoproteins is still unclear. To clarify the relationship between glycosyltransferases and sugar chain branch structure, we estimated six glycosyltransferases' activities (N-acetylglucosaminyltransferase (GlcNAcTase)-I, -II, -III, -IV, -V, and β-1,4-galactosyltransferase (GalT)) which control the branch formation on asparagine (Asn)-linked sugar chains in 18 human cancer cell lines derived from several tissues. To visualize the balance of glycosyltransferase activity associated with each cell line, we expressed the relative glycosyltransferase activity in comparison to the average activity among the cell lines. These cell lines were classified into five groups according to their relative glycosyltransferase balance and were termed GlcNAcTase-I/-II, GlcNAcTase-III, GlcNAcTase-IV, GlcNAcTase-V, and GalT. We also characterized the structures of Asn-linked sugar chains on the cell surface of representative cell lines of each group. The branching structure of cell surface sugar chains roughly corresponded to the glycosyltransferase balance. This finding suggests that, for the sugar chain structure remodeling of glycoproteins, attention should be focused on the glycosyltransferase balance of host cells before introducing exogenous glycosyltransferases or down-regulating the activity of intrinsic glycosyltransferases.
Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been ...analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation. Published in 2004. PUBLICATION ABSTRACT
Bovine zone pellucida (ZP) glycoproteins from ovarian egg emerged as three bands with molecular mass of 78 kDa, 64 kDa and 21 kDa in SDS-PAGE under reducing conditions. Endo-beta-galactosidase ...(EbetaG) digestion of the glycoproteins yielded five products with molecular mass of 76 kDa (EbetaG-76), 68 kDa (EbetaG-68), 63 kDa (EbetaG-63), 47 kDa (EbetaG-47) and 21 kDa (EbetaG-21) under the same conditions. The N-terminal amino acid sequences of EbetaG-76 and EbetaG-21 were identical. This fact together with the results of diagonal SDS-PAGE indicated that EbetaG-21 (N-terminal region) is linked to EbetaG-63 (C-terminal region) through disulfide bond to form EbetaG-76. Immunoblot analysis using anti-pig ZP protein antibodies revealed that bovine EbetaG-76, EbetaG-68 and EbetaG-47 correspond to pig PZP2, PZP3 alpha and PZP3 beta glycoproteins, respectively. The EbetaG-76 and EbetaG-68 components were shown to be specifically cleaved during fertilization.
The three glycoproteins of pig zona pellucida (ZPA, ZPB and ZPC) can be separated into ZPA and a mixture of ZPB/ZPC by gel‐filtration HPLC. We have shown previously that the neutral complex‐type ...N‐linked carbohydrate chains obtained from ZPB/ZPC possess sperm‐binding activity. Intact ZPB and ZPC cannot be separated from each other unless acidic N‐acetyllactosamine regions of their carbohydrate chains are removed by endo‐β‐galactosidase digestion. The endo‐β‐galactosidase−digested ZPB retains the sperm‐binding activity. Recently, we have reported that N‐linked carbohydrate chains of N‐terminal fragment (residues 137−247) obtained from endo‐β‐galactosidase−digested ZPB are involved mainly in sperm binding Yonezawa, N., Mitsui, S., Kudo, K. & Nakano, M. (1997) Eur. J. Biochem. 248, 86−92. In this study, we separated the intact neutral N‐linked chains from the ZPB/ZPC mixture into diantennary chains and triantennary and tetraantennary chains by affinity chromatography on Concanavalia ensiformis agglutinin. An in vitro competition assay revealed that triantennary and tetraantennary chains possess a sperm‐binding activity stronger than that of diantennary chains. Three glycopeptides, having one Asn residue to which the carbohydrate chain is linked, were obtained by lysyl endopeptidase digestion of the heat‐solubilized zonae containing intact ZPB and lysyl endopeptidase and chymotrypsin A digestion of endo‐β‐galactosidase−digested ZPB. From sugar‐mapping analysis of the carbohydrate chains from these glycopeptides and comparison with the carbohydrate structures of the main intact neutral N‐linked chains of ZPB/ZPC, the triantennary and tetraantennary chains were shown to be localized mainly at Asn220 of ZPB, and diantennary chains were present on all the three potential residues (Asn203, Asn220 and Asn333). These results suggest that the carbohydrate chains linked to Asn220 of ZPB participate predominantly in sperm‐egg binding.
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